ABSTRACT Enterococci are one of the most recalcitrant hospital-associated pathogens due to resistance to many antibiotics used in clinical practice with some untreatable infections occurring in immunocompromised individuals. The CDC conservatively estimates that vancomycin-resistant enterococci (VRE) are associated with 20,000 infections and 1,300 deaths per year in the US alone. VRE typically affect patients who have multiple comorbidities or with important compromise of the immune system, including solid organ transplant patients and those with hematological malignancies, among others. Surprisingly, despite the frequent occurrence of VRE in these vulnerable populations, prospective studies assessing the actual clinical impact of infections due to these organisms are scarce, limiting the availability of clinical information to guide treatment for these recalcitrant infections. Furthermore, the paucity of reliable antimicrobial options to treat severe disease is of major concern. Indeed, enterococci have developed resistance to virtually all anti-enterococcal antibiotics available in clinical practice. Currently, the lipopetide antibiotic daptomycin (DAP) has become the first-line therapy due to its bactericidal activity and safety profile, despite lacking FDA approval for this indication. However, uncertainties on the performance of MIC testing, DAP breakpoint and appropriate dosing for enterococci are major limitations for using this antibiotic against VRE. Additionally, resistance and tolerance to DAP readily emerge during therapy via chromosomal mutations in genes encoding the LiaFSR system, a three component regulatory system that controls the enterococcal cell membrane stress response. In order to fill this major vacuum in knowledge and optimize the management of enterococcal bacteremia, we have assembled the VENOUS cohort (Vancomycin-Resistant ENterococci OUtcomes Study), a unique prospective cohort of patients with enterococcal bacteremia currently recruiting in 17 hospitals in the USA (7 cities) and additional 4 hospitals in South America (n=2) and Europe (n=2). Our overarching hypothesis is that a deep understanding of the clinical and microbiological aspects of VRE bloodstream infections and dynamics of the population structure of infecting isolates is crucial to help design novel diagnostic approaches and treatment regimens to improve the outcomes of these difficult-to-treat infections. Using the VENOUS study we propose to i) characterize the clinical impact of VRE bacteremia, ii) dissect the population structure of VRE causing bloodstream infections and, iii) develop a new minimal inhibitory concentration (MIC)-independent diagnostic test to assess DAP susceptibility, seeking to guide clinicians with a novel tool to allow accurate identification of DAP-susceptible isolates and improve the use of DAP and combination with ?-lactams against these organisms. The results of this proposal are likely to provide much needed and robust data to optimize the treatment of VRE infections, deliver the necessary information to plan future interventional studies and develop innovative diagnostic approaches to revolutionize the management of these life-threatening infections.